AmpC b-Lactamases

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AmpC b-Lactamases their detection

• David Livermore
• Health Protection Agency,
• Colindale, London

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b-Lactamase-stable cephs
S
CONH
N
O
R
COOH
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b-Lactamase classes
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AmpC enzymes

• Mol wt. c. 40,000 alkaline pI
• Hydrolytic activity
• 1st gen cephs rapid
• 2/3 ceph cephs slow but kinetically efficient
• 4-gen cephs slow, kinetically inefficient
• Carbapenems nearly stable. not quite!
• Not inhibited by clavulanate poorly inhibited by
  sulphones

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AmpC ?-lactamases

• Basal in
• E. coli shigellae
• Inducible in
• Enterobacter spp.
• C. freundii
• M. morganii
• Serratia spp.
• P. aeruginosa
• 2nd, 3rd gen cephs
• Labile, but weak inducers, select derepressed
  mutants

Derepressed
Inducible
Amt ?-lactamase
? -lactam
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Induction of AmpC

• Cell wall constantly re-cycled
• Yields disaccharide tri-peptides (DTPs)
• Absorbed by AmpG
• Cleaved by AmpD N-acetyl-muramyl-L-alanine
  amidase
• Excess DTPs bind AmpR, activating ampC

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Uninduced AmpC

• Wall fragments recycled by AmpD
• AmpR in repressor conformation
• ampC (?-lactamase gene) NOT expressed

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Induced AmpC
?-lactamase
AmpD
ampC
ampR
ampD

• More recycling AmpD overwhelmed
• Wall fragments convert AmpR to activator
• ampC (?-lactamase gene) expressed

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Derepressed AmpC
?-lactamase
ampC
ampR
ampD

• ampD inactivated by mutation
• AmpR constantly converted to activator
• ampC hyper-expressed

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Strong weak inducers
Strong inducer Weak inducer
Labile 1st gen cephs Ampicillin 3rd gen cephs Piperacillin Aztreonam
Stable Imipenem (Temocillin) (Meropenem)
Strong inducer induces below MIC, weak doesnt
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MICs (mg/L) for E. cloacaeAmpC mutants
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MICs (mg/L) for P. aeruginosa AmpC mutants
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What selects derepression?

• Strong inducers e.g. imipenem

No! derepressed no more R than inducible
All weak inducers?
No- Not if they are stable
LABILE weak inducers?
Yes! Derepressed are R, whilst inducible cells
are S, so derepressed are selected
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Over-run by mutants
Mutant emerges randomly

• Derepression occurs in 1 cell / 107
• Confers resistance to 3-gen cephs
• Overnight, one cell can give 109 progeny
• Selection in therapy can cause Rx failure...

Sensitive cells killed by antibiotic
Mutants progeny overrun
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Initial isolation of 3rd-gen cephR Enterobacter,
prior Rx
Chow et al. Ann Intern Med 1991, 115, 585-90
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Selecting Enterobacter R to 3rd gen cephs during
Rx
Kaye et al. AAC 2001, 45, 2628 Cosgrove et al.
Arch Intern Med 2002, 162, 185
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Plasmidic AmpC enzymes

• Escaping from enterobacterial and Aeromonas
  chromosomes
• Many good reports since 1991
• CMY, MOX, FOX, LAT, DHA, ACT BIL enzymes

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Sources of plasmid AmpC
Class Source Examples
CIT C. freundii CMY-2 to 7 LAT-1,3,4
ENT Enterobacter spp. ACT-1 MIR-1
FOX Aeromonas spp. FOX-1 to -5
MOX Aeromonas spp. MOX-1,-2 CMY-1,7 8
DHA M morganii DHA-1, -2
ACC H. alvei ACC-1
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Plasmid AmpC
Jenks et al. 1995. J Antimicrob Chemother 35,
235-6.
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Enzymes in 1127 cephR isolates from 16 labs in S
England, 2004
Potz et al., JAC, 2006 in press
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Prevalence of mechanisms BSAC bacteraemia
surveillance, 2005
http//www.bsacsurv.org
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Inducible AmpC
Cefoxitin
But mutational derepression is the problem, not
induction Better predict risk from species I/D
Ceftazidime
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Suspect derepressed / plasmid AmpC if

• Resistant 3-gen cephs, NOT cefepime cefpirome
• Resistant to cefoxitin (but more ESBL producers
  R, too, nowadays)
• No ceph/clav synergy

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Geometric mean MICs (mg/L) AmpC producers 2004
London SE survey
E. coli AmpC E. coli ESBL Enterobacter AmpC Enterobacter ESBL
Cefotaxime 12 228 72 49
Ceftazidime 18 39 36 81
Cefepime 0.38 39 0.91 4.4
Cefpirome 0.57 53 2.4 10
Cefuroxime 35 gt64 gt64 gt64
Cefoxitin 51 17 gt64 51
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Some wrinkles

• AmpC-derepressed M. morganii are S to pip/tazo
• AmpC derepressed Serratia are S to ceftazidime
• Cefoxitin R an unreliable marker for Providencia,
  Morganella Serratia spp.
• Inducible derepressed strains may appear I or S
• AmpC derepressed P. aeruginosa tend to be S to
  carbenicillin / efflux mutants are R
• Life complicated if theres an ESBL with the AmpC

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Confirmatory tests for AmpC

• Seek cefotaxime/cloxacillin synergy
• Cefotaxime MIC 100 mg/l cloxacillin
• Zones of cefotaxime 30 mg discs on agar 100
  mg/L cloxacillin
• No agreed interpretive standards
• Can also use phenylboronic acid as inhibitor
• DHA enzymes inducible especially difficult to
  detect

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Cefotaxime combinations vs. AmpC E. coli London
SE survey
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Cefotaxime combinations vs. AmpC E. coli London
SE survey
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Cefotaxime / cloxacillin tests for AmpC
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3-D test for AmpCs
Plate seeded with cefoxitin S indicator strain
Cut cross in agar, heavily inoculated with test
strain
Cefoxitin disc
Looks for distortion where cross intersects the
cefoxitin zone
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Clover leaf (3 dimensional) test for AmpC
Test strain E. cloacae, AmpC derepressed Indicato
r E. coli NCTC10418 Disc Cefoxitin 30 mg
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Clover leaf (3 dimensional) test for
cephalosporinase
Test strain E. cloacae, AmpC derepressed Indicato
r E. coli NCTC10418 Disc Cefotaxime 30 mg
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Phenyl boronic acid for detection of plasmid AmpC
Expansion (mm) of FOX 30 zone with 400 mg phenyl boronic acid Fold MIC reduction for cefoxitin 400 mg/L phenyl boronic acid
Kleb MOX-1 12 128
E. coli LAT-2 12 64
Kleb DHA-1 14 128
Kleb DHA-2 13 64
E. coli ACC-1 4 4
Kleb ACT-1 13 64
ALL ESBL ve lt2 lt2
Coudron JCM 2005 43 4163
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Disc tests for AmpC
60 E. coli Klebsiella cefoxitin MICs reduced gt4-fold by 100 mg/L cloxacillin with gt5 mm zone expansion
Cefoxitin cloxacillin 100 mg 86
Cefoxitin BZB 64 mg 89
Cefpodoxime BZB 64 mg 97
Cefpodoxime clav BZB 64 mg 100
BZB benzo(b)thiophene-2-boronic acid
Brenwald et al., JAC 2005, 56, 600
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Cefoxitin R isolates in phenyl boronic acid /
cefoxitin tests
3-D 3-D Phenyl boronic, disc Phenyl boronic, disc Phenyl boronic MIC Phenyl boronic MIC
- - -
K. pneumoniae 106 16 110 12 110 12
K. oxytoca 4 2 5 1 5 1
E. coli 22 93 40 75 39 76
P. mirabilis 24 4 25 3 26 2
Coudron JCM 2005 43 4163
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Multiplex detection of plasmid AmpC genes
Method of Perez-Perez Hanson JCM 2002, 40, 2153
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AmpC hyperproducers options

• Active
• Carbapenems
• Temocillin
• 4-gen cephs

• Not active
• Penicillins except temocillin
• Inhibitor combinations
• 1, 2, 3-gen cephs
• Aztreonam

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AmpC Summary

• Mutant selection the problem, not induction
• Selection mostly in therapy with 3-gen cephs
• AmpC types spreading to plasmids
• Suspect AmpC if
• R 3rd, not 4th gen cephs cefoxitin R, no
  ceph/clav synergy
• Confirmatory tests poorly standardised, exploit
  synergy with cloxacillin or phenyl boronic acids